Glycosylation is one of the most common post-translational protein modifications in eukaryotic systems. It has been estimated that over half of all mammalian proteins are glycosylated at some point during their existence and virtually all membrane and secreted proteins are glycosylated. Glycosylation is a non-template-driven process and is believed to introduce the high level of variability necessary for complex processes in higher organisms. In addition to participating in key macromolecular interactions, glycans have been shown to contribute to protein folding, trafficking, and stability.
N-glycans are linked to the protein backbone via asparagine residues that are part of the tripeptide sequences Asn-X-Ser or Asn-X-Thr, with X being any amino acid except proline. Depending on the terminal sugar residues, N-glycans are classified into complex, high-mannose, and hybrid N-glycans. This classification is based on the common pentasaccharide motif shared by most N-glycans. O-glycans are linked via serine or threonine residues to the protein. There are a number of O-glycan core structures, with the most common being Core 1, Core 2, Core 3, and Core 4.
Numerous diseases are known to involve acquired changes in glycosylation and/or in the recognition of glycans. For example, altered glycosylation is a universal feature of cancer cells and some glycan structures are well-known markers for tumours and tumour progression. As a result, methods for the comprehensive analysis of protein glycosylation and glycan composition are of interest to the scientific community.
For most glycol-profiling methods, the glycans are removed from the protein either by hydrazinolysis or treatment with a specific peptide glycosidase (e.g. PNGase F). Owing to its high sensitivity at low concentrations, mass spectrometry is often used in the analysis of the resulting complex mixtures. However, the signal intensity of particular analytes is dependent, amongst many other factors, on the physical properties (likelihood of ionisation, tendency to fragment, etc.) of the analyte, making any relative quantification, and sometimes even identification, very difficult.
Identification of glycans common to two samples and their relative quantification may be facilitated by use of derivatisation of the glycan mixtures to incorporate isotopic tags. The two samples are labelled with the light and heavy form of the labelling reagent and then mixed prior to analysis using mass spectrometry. Derivatisation to incorporate isotopic tags into glycan mixtures isolated from glycoproteins has been accomplished using permethylation techniques or glycan reductive isotope labelling, in which the tag is introduced using reductive amination (Atwood, 2007; Bowman, 2007, 2010; Botelho, 2008; Hitchcock, 2006; Hsu, 2006; Kang, 2007; Lawrence, 2008; Ridlova, 2008; Yuan, 2005; Zhang, 2003).
Reductive amination typically occurs at the reducing end of the glycans and may use isotopically-labelled aniline, aminopyridine or anthranilic acid. For example, Xia et al. (2009) have demonstrated the use of isotopically-labelled aniline tags to compare the differences in mixtures of glycans released from human and mouse sera. The glycans were released by PNGase F, then the resulting mixtures separately derivatised by reductive amination with 12C6-aniline or 13C6-aniline. By analysing an equimolar combination of the 12C6-aniline-derivatised mixture of glycans from mouse serum and the 13C6-aniline-derivatised mixture of glycans from human serum, the authors reported that they were able to identify paired mass peaks separated by a mass difference of 6 Da and assign plausible structures for glycans common to both samples. The authors reported that a comparison of the relative intensities of these peaks enabled a determination of the amount of a particular common glycan present in one sample compared to the other.
However, these methods provide only semi-quantitative results. Furthermore, the results are affected by the reproducibility of the tagging procedures and problems caused by side reactions, oxidative degradation and “peeling reactions” (which may occur due to certain reaction conditions in aqueous solutions), and important functionalization may be lost during the derivatisation step.
Isotopic tags have also been used in proteomics. Breidenbach et al. (2012) have demonstrated the metabolic incorporation of isotopically-labelled GlcNAc into yeast N-glycans using filter aided sample preparation methodology. A GlcNAc isomix was used comprising natural isotope abundance GlcNAc, 13C2-GlcNAc and 13C415N1-GlcNAc in a 1:2:1 ratio to mimic the dibromide isotope triplet pattern. The resulting glycol conjugates containing the isomix underwent FASP digestion and EndoH deglycosylation and were analysed using an automated isotopic envelope pattern search (in LC-MS/MS experiments) to facilitate glycoside identification. The method enabled the authors to place fragmentation priority on glycopeptides ions regardless of their relative intensities to other ions in the sample.
There exists an unmet need for improved methods for rapidly and easily analysing the content of released glycan mixtures, and in particular one which does not suffer from the disadvantages of the described prior art.